Background incidence rates of health outcomes in populations at risk for Lyme disease using US administrative claims data.

BACKGROUND: Background incidence rates (IRs) of health outcomes in Lyme disease endemic regions are useful to contextualize events reported during Lyme disease vaccine clinical trials or post-marketing. The objective of this study was to estimate and compare IRs of health outcomes in Lyme disease endemic versus non-endemic regions in the US during pre-COVID and COVID era timeframes. METHODS: IQVIA PharMetrics® Plus commercial claims database was used to estimate IRs of 64 outcomes relevant to vaccine safety monitoring in the US during January 1, 2017-December 31, 2019 and January 1, 2020-December 31, 2021. Analyses included all individuals aged ≥ 2 years with ≥ 1 year of continuous enrollment. Outcomes were defined by International Classification of Diseases Clinical Modification, 10th Revision (ICD-10-CM) diagnosis codes. IRs and 95 % confidence intervals (CIs) were calculated for each outcome and compared between endemic vs. non-endemic regions, and pre-COVID vs. COVID era using IR ratios (IRR). RESULTS: The study population included 8.7 million (M) in endemic and 27.8 M in non-endemic regions. Mean age and sex were similar in endemic and non-endemic regions. In both study periods, the IRs were statistically higher in endemic regions for anaphylaxis, meningoencephalitis, myocarditis/pericarditis, and rash (including erythema migrans) as compared with non-endemic regions. Conversely, significantly lower IRs were observed in endemic regions for acute kidney injury, disseminated intravascular coagulation, heart failure, myelitis, myopathies, and systemic lupus erythematosus in both study periods. Most outcomes were statistically less frequent during the COVID-era. CONCLUSION: This study identified potential differences between Lyme endemic and non-endemic regions of the US in background IRs of health conditions during pre-COVID and COVID era timeframes to inform Lyme disease vaccine safety monitoring. These regional and temporal differences in background IRs should be considered when contextualizing possible safety signals in clinical trials and post-marketing of a vaccine targeted at Lyme disease prevention.

The role of early functional neuroimaging in predicting neurodevelopmental outcomes in neonatal encephalopathy.

Reliably assessing the early neurodevelopmental outcomes in infants with neonatal encephalopathy (NE) is of utmost importance to advise parents and implement early and personalized interventions. We aimed to evaluate the accuracy of neuroimaging modalities, including functional magnetic resonance imaging (fMRI) in predicting neurodevelopmental outcomes in NE. Eighteen newborns with NE due to presumed perinatal asphyxia (PA) were included in the study, 16 of whom underwent therapeutic hypothermia. Structural magnetic resonance imaging (MRI), and fMRI during passive visual, auditory, and sensorimotor stimulation were acquired between the 10th and 14th day of age. Clinical follow-up protocol included visual and auditory evoked potentials and a detailed neurodevelopmental evaluation at 12 and 18 months of age. Infants were divided according to sensory and neurodevelopmental outcome: severe, moderate disability, or normal. Structural MRI findings were the best predictor of severe disability with an AUC close to 1.0. There were no good predictors to discriminate between moderate disability versus normal outcome. Nevertheless, structural MRI measures showed a significant correlation with the scores of neurodevelopmental assessments. During sensorimotor stimulation, the fMRI signal in the right hemisphere had an AUC of 0.9 to predict absence of cerebral palsy (CP). fMRI measures during auditory and visual stimulation did not predict sensorineural hearing loss or cerebral visual impairment. CONCLUSION: In addition to structural MRI, fMRI with sensorimotor stimulation may open the gate to improve the knowledge of neurodevelopmental/motor prognosis if proven in a larger cohort of newborns with NE. WHAT IS KNOWN: • Establishing an early, accurate neurodevelopmental prognosis in neonatal encephalopathy remains challenging. • Although structural MRI has a central role in neonatal encephalopathy, advanced MRI modalities are gradually being explored to optimize neurodevelopmental outcome knowledge. WHAT IS NEW: • Newborns who later developed cerebral palsy had a trend towards lower fMRI measures in the right sensorimotor area during sensorimotor stimulation. • These preliminary fMRI results may improve future early delineation of motor prognosis in neonatal encephalopathy.

Laser Microdissection of Woody and Suberized Plant Tissues for RNA-Seq Analysis.

An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.

Molecular Imprinted Polymers on Microneedle Arrays for Point of Care Transdermal Sampling and Sensing of Inflammatory Biomarkers.

The skin interstitial fluid (ISF) contains biomarkers that complement other biofluids such as blood, sweat, saliva, and urine. It can be sampled in a minimally invasive manner and used either for point of care testing or real time, continuous monitoring of analytes, the latter using microneedle arrays. The analytes present in the skin ISF are indicative of both systemic and local (i.e., skin) physiology. In this paper, we describe combining microneedle technology with molecularly imprinted polymers to demonstrate the potential of transdermal electrochemical sensing. The molecularly imprinted polymer employed here is easy to produce; it can be thought of as plastic antibody. Its synthesis is scalable, and the resulting sensor has a short measurement time (6 min), with high accuracy and a low limit of detection. It provides the requisite specificity to detect the proinflammatory cytokine IL-6. IL-6 is present in the skin ISF with other cytokines and is implicated in many clinical states including neurodegenerative diseases and fatal pneumonia from SARSCoV 2. The ability to mass produce microneedle arrays and plastic antibodies will allow for low-cost transdermal sensing devices. The transdermal sensors were able to detect IL-6 at concentrations as low as 1 pg/mL in artificial skin ISF, indicating its utility for routine point of care, bloodless measurements in simpler settings, worldwide.

Development of colorimetric cellulose-based test-strip for the rapid detection of antibodies against SARS-CoV2 virus.

Given the pandemic situation, there is an urgent need for an accurate test to monitor antibodies anti-SARS-CoV-2, providing crucial epidemiological and clinical information to monitor the evolution of coronavirus disease in 2019 (COVID-19) and to stratify the immunized and asymptomatic population. Therefore, this paper describes a new cellulose-based test strip for rapid and cost-effective quantitative detection of antibodies to SARS-CoV2 virus by colorimetric transduction. For this purpose, Whatman paper was chemically modified with sodium metaperiodate to introduce aldehyde groups on its surface. Subsequently, the spike protein of the virus is covalently bound by forming an imine group. The chemical control of cellulose paper modification was evaluated by Fourier transform infrared spectroscopy, thermogravimetry and contact angle analysis. Colorimetric detection of the antibodies was performed by a conventional staining method using Ponceau S solution as the dye. Color analysis was performed after image acquisition with a smartphone using Image J software. The color intensity varied linearly with the logarithm of the anti-S concentration (from 10 ng/mL to 1 µg/mL) in 500-fold diluted serum samples when plotted against the green coordinate extracted from digital images. The test strip was selective in the presence of nucleocapsid antibodies, urea, glucose, and bovine serum albumin with less than 15% interference, and detection of antibodies in human serum was successfully performed. Overall, this is a simple and affordable design that can be readily used for mass population screening and does not require sophisticated equipment or qualified personnel. Supplementary Information: The online version contains supplementary material available at 10.1007/s10570-022-04808-y.

Water Deficit Timing Differentially Affects Physiological Responses of Grapevines Infected with Lasiodiplodia theobromae.

Diseases and climate change are major factors limiting grape productivity and fruit marketability. Lasiodiplodia theobromae is a fungus of the family Botryosphaeriaceae that causes Botryosphaeria dieback of grapevine worldwide. Abiotic stress may change host vitality and impact susceptibility to the pathogen and/or change the pathogen's life cycle. However, the interaction between both stress drivers is poorly understood for woody plants. We addressed the hypothesis that distinct morpho-physiological and biochemical responses are induced in grapevine (Vitis vinifera)-L. theobromae interactions depending on when water deficits are imposed. Grapevines were submitted to water deficit either before or after fungus inoculation. Water deficit led to the reduction of the net photosynthetic rate, stomatal conductance, and transpiration rate, and increased the abscisic acid concentration regardless of fungal inoculation. L. theobromae inoculation before water deficit reduced plant survival by 50% and resulted in the accumulation of jasmonic acid and reductions in malondialdehyde levels. Conversely, grapevines inoculated after water deficit showed an increase in proline and malondialdehyde content and all plants survived. Overall, grapevines responded differently to the primary stress encountered, with consequences in their physiological responses. This study reinforces the importance of exploring the complex water deficit timing × disease interaction and the underlying physiological responses involved in grapevine performance.

Colorimetric cellulose-based test-strip for rapid detection of amyloid ß-42.

An innovative sensing assay is described for point-of-care (PoC) quantification of a biomarker of Alzheimer's disease, amyloid ß-42 (Aß-42). This device is based on a cellulose paper-dye test strip platform in which the corresponding detection layer is integrated by applying a molecularly imprinted polymer (MIP) to the cellulose paper surface. Briefly, the cellulose paper is chemically modified with a silane to subsequently apply the MIP detection layer. The imprinting process is confirmed by the parallel preparation of a control material, namely a non-imprinted polymer (NIP). The chemical changes of the surface were evaluated by Fourier transform infrared spectroscopy (FTIR), contact angle, and thermogravimetric analysis (TG). Proteins and peptides can be quantified by conventional staining methods. For this purpose, Coomassie blue (CB) was used as a staining dye for the detection and quantification of Aß-42. Quantitative determination is made possible by taking a photograph and applying an appropriate mathematical treatment to the color coordinates provided by the ImageJ program. The MIP shows a linear range between 1.0 ng/mL and 10 µg/mL and a detection limit of 0.71 ng/mL. Overall, this cellulose-based assay is suitable for the detection of peptides or proteins in a sample by visual comparison of color change. The test strip provides a simple, instrument-free, and cost-effective method with high chemical stability, capable of detecting very small amounts of peptides or proteins in a sample, and can be used for the detection of any (bio)molecule of interest.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity.

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Successful metabolic control in diabetes type 1 depends on individual neuroeconomic and health risk-taking decision endophenotypes: a new target in personalized care.

BACKGROUND: Neurobehavioral decision profiles have often been neglected in chronic diseases despite their direct impact on major public health issues such as treatment adherence. This remains a major concern in diabetes, despite intensive efforts and public awareness initiatives regarding its complications. We hypothesized that high rates of low adherence are related to risk-taking profiles associated with decision-making phenotypes. If this hypothesis is correct, it should be possible to define these endophenotypes independently based both on dynamic measures of metabolic control (HbA1C) and multidimensional behavioral profiles. METHODS: In this study, 91 participants with early-stage type 1 diabetes fulfilled a battery of self-reported real-world risk behaviors and they performed an experimental task, the Balloon Analogue Risk Task (BART). RESULTS: K-means and two-step cluster analysis suggest a two-cluster solution providing information of distinct decision profiles (concerning multiple domains of risk-taking behavior) which almost perfectly match the biological partition, based on the division between stable or improving metabolic control (MC, N = 49) v. unstably high or deteriorating states (NoMC, N = 42). This surprising dichotomy of behavioral phenotypes predicted by the dynamics of HbA1C was further corroborated by standard statistical testing. Finally, the BART game enabled to identify groups differences in feedback learning and consequent behavioral choices under ambiguity, showing distinct group choice behavioral patterns. CONCLUSIONS: These findings suggest that distinct biobehavioral endophenotypes can be related to the success of metabolic control. These findings also have strong implications for programs to improve patient adherence, directly addressing risk-taking profiles.

Temporal physiological response of pine to Fusarium circinatum infection is dependent on host susceptibility level: the role of ABA catabolism.

Pine pitch canker (PPC), caused by Fusarium circinatum Nirenberg and O'Donnell, represents an important threat to conifer forests worldwide, being associated with significant economic losses. Although essential to develop disease mitigation strategies, little research focused on host susceptibility/resistance mechanisms has been conducted. We aimed to explore the response of a highly susceptible (Pinus radiata D. Don) and a relatively resistant (Pinus pinea L.) species to F. circinatum infection at different stages of infection. Morpho-physiological, hormonal and oxidative stress-related changes were assessed for each pine species and sampling point. Most of the changes found occurred in symptomatic P. radiata, for which an increased susceptibility to photoinhibition was detected together with decreased superoxide dismutase activity. Abscisic acid catabolism was activated by F. circinatum inoculation in both pine species, leading to the accumulation of the inactive dihydrophaseic acid in P. radiata and of the less-active phaseic acid in P. pinea. Hormone confocal analysis revealed that this strategy may be of particular importance at 6 d.p.i. in P. pinea, which together with photosynthesis maintenance to fuel defense mechanism, could in part explain the species resistance to PPC. These results are of great interest for the development of hormone-based breeding strategies or for the use of hormone application as inducers of resistance to F. circinatum infection.

In-depth analysis of the Quercus suber metabolome under drought stress and recovery reveals potential key metabolic players.

Cork oak (Quercus suber L.) is a species of ecological, social and economic importance in the Mediterranean region. Given its xerophytic adaptability, the study of cork oak's response to drought stress conditions may provide important data in the global scenario of climate change. The mechanisms behind cork oak's adaptation to drought conditions can inform the design and development of tools to better manage this species under the changing climate patterns. Metabolomics is one of the most promising omics layers to capture a snapshot of a particular physiological state and to identify putative biomarkers of stress tolerance. Drastic changes were observed in the leaf metabolome of Q. suber between the different experimental conditions, namely at the beginning of the drought stress treatment, after one month under drought and post rehydration. All experimental treatments were analyzed through sPLS to inspect for global changes and stress and rehydration responses were analyzed independently for specific alterations. This allowed a more in-depth study and a search for biomarkers specific to a given hydric treatment. The metabolome analyses showed changes in both primary and secondary metabolism, but highlighted the role of secondary metabolism. In addition, a compound-specific response was observed in stress and rehydration. Key compounds such as L-phenylalanine and epigallocatechin 3-gallate were identified in relation to early drought response, terpenoid leonuridine and the flavonoid glycoside (-)-epicatechin-3'-O-glucuronide in long-term drought response, and flavone isoscoparine was identified in relation to the recovery process. The results here obtained provide novel insights into the biology of cork oak, highlighting pathways and metabolites potentially involved in the response of this species during drought and recovery that may be essential for its adaptation to long periods of drought. It is expected that this knowledge can encourage further functional studies in order to validate potential biomarkers of drought and recovery that maybe used to support decision-making in cork oak breeding programs.

Language Predictors in Autism Spectrum Disorder: Insights from Neurodevelopmental Profile in a Longitudinal Perspective.

Language outcome in individuals with autism spectrum disorder (ASD) is predicted by early developmental milestones and cognitive abilities. The development and acquisition of expressive language (particularly the onset of first phrases) is a relevant clinical milestone by school age, since its early presentation is associated to better long-term life outcomes and to lower core clinical severity of ASD. Focusing on predictors of language in ASD children, a number of outstanding questions remain to be answered, namely, whether there are differences in the early key neurodevelopmental abilities and whether those differences in a specific period of time might predict verbal development and acquisition of expressive language. We aim to understand how the neurodevelopmental profile of ASD children evolves from the preschool to the school age and if and which subarea can better predict acquisition of expressive language. Children with ASD (N = 205) were evaluated with a structured assessment of neurodevelopment in two different age periods: 1) preschool period (mean age four years) and 2) reassessment in the school period (mean age seven years). Our findings demonstrate that in nonverbal preschool children with ASD normal or near normal Performance Developmental Quotient (superior to 73.5) evaluated at preschool age is a good predictor of later language development in ASD, which has important implications for intervention programs targeting this population and family information.

A Gradient of Hippocampal Inputs to the Medial Mesocortex.

Memory-guided decisions depend on complex interactions between the hippocampus (HIPP) and medial mesocortical (MMC) regions, including the anterior cingulate (CG) and retrosplenial (RSC). The functional circuitry underlying these interactions is unclear. Using anatomy, electrophysiology, and optogenetics, we show that such circuitry is characterized by a functional-anatomical gradient. While the CG receives hippocampal excitatory projections originated in CA1 stratum pyramidale, the RSC additionally receives long-range inhibitory inputs from radiatum and lacunosum-moleculare. Such hippocampal projections establish bona fide synapses, with the RSC densely targeted on its superficial layers L1-L3 by a combination of inhibitory and excitatory synapses. We show that the MMC is targeted by dorsal-intermediate CA1 (diCA1) axons following a caudorostral gradient in which a dense, dual (excitatory/inhibitory), layer-specific projection is progressively converted in a sparse, excitatory, and diffuse projection. This gradient is reflected in higher oscillatory synchronicity between the HIPP and RSC in the awake-behaving animal, compatible with their known functional proximity and contrasting with that found in the CG.

Embryonic development, locomotor behavior, biochemical, and epigenetic effects of the pharmaceutical drugs paracetamol and ciprofloxacin in larvae and embryos of Danio rerio when exposed to environmental realistic levels of both drugs.

For several years, the scientific community has been concerned about the presence of pharmaceuticals in the wild, since these compounds may have unpredictable deleterious effects on living organisms. Two examples of widely used pharmaceuticals that are present in the environment are paracetamol and ciprofloxacin. Despite their common presence in the aquatic environment due to their poor removal by sewage treatment plants, knowledge concerning their putative toxic effects is still scarce. This work aimed to characterize the effects of paracetamol (0.005, 0.025, 0.125, 0.625, and 3.125 mg/L) and ciprofloxacin (0.005, 0.013, 0.031, 0.078, 0.195, and 0.488 µg/L) in zebrafish embryos and larvae, exposed to environmentally relevant levels, close to the real concentrations of these pharmaceuticals in surface waters and effluents. The adopted toxic end points were developmental, a behavioral parameter (total swimming time), and a biomarker-based approach (quantification of the activities of catalase, glutathione-S-transferase, cholinesterases, glutathione peroxidase, and lipid peroxidation levels) combined with epigenetic analysis (immunohistochemical detection of 5-methylcytidine). Exposure to paracetamol had effects on all of the adopted toxic end points; however, ciprofloxacin only caused effects on behavioral tests and alterations in biomarkers. It is possible to ascertain the occurrence of oxidative stress following exposure to both drugs, which was more evident regarding paracetamol, an effect that may be related to the observed epigenetic modifications.

mTOR Signaling Pathway Regulates Sperm Quality in Older Men.

Understanding how age affects fertility becomes increasingly relevant as couples delay childbearing toward later stages of their lives. While the influence of maternal age on fertility is well established, the impact of paternal age is poorly characterized. Thus, this study aimed to understand the molecular mechanisms responsible for age-dependent decline in spermatozoa quality. To attain it, we evaluated the impact of male age on the activity of signaling proteins in two distinct spermatozoa populations: total spermatozoa fraction and highly motile/viable fraction. In older men, we observed an inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) in the highly viable spermatozoa population. On the contrary, when considering the entire spermatozoa population (including defective/immotile/apoptotic cells) our findings support an active mTORC1 signaling pathway in older men. Additionally, total spermatozoa fractions of older men presented increased levels of apoptotic/stress markers (e.g., cellular tumor antigen p53-TP53) and mitogen-activated protein kinases (MAPKs) activity. Moreover, we established that the levels of most signaling proteins analyzed were consistently and significantly altered in men more than 27 years of age. This study was the first to associate the mTOR signaling pathway with the age impact on spermatozoa quality. Additionally, we constructed a network of the sperm proteins associated with male aging, identifying TP53 as a central player in spermatozoa aging.

Pinus Susceptibility to Pitch Canker Triggers Specific Physiological Responses in Symptomatic Plants: An Integrated Approach.

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is an emergent and still understudied risk that threatens Pinus forests worldwide, with potential production and sustainability losses. In order to explore the response of pine species with distinct levels of susceptibility to PPC, we investigated changes in physiology, hormones, specific gene transcripts, and primary metabolism occurring in symptomatic Pinus pinea, Pinus pinaster, and Pinus radiata upon inoculation with F. circinatum. Pinus radiata and P. pinaster exhibiting high and intermediate susceptibility to PPC, respectively, suffered changes in plant water status and photosynthetic impairment. This was associated with sink metabolism induction, a general accumulation of amino acids and overexpression of pathogenesis-related genes. On the other hand, P. pinea exhibited the greatest resistance to PPC and stomatal opening, transpiration increase, and glycerol accumulation were observed in inoculated plants. A stronger induction of pyruvate decarboxylase transcripts and differential hormones regulation were also found for inoculated P. pinea in comparison with the susceptible Pinus species studied. The specific physiological changes reported herein are the first steps to understand the complex Pinus-Fusarium interaction and create tools for the selection of resistant genotypes thus contributing to disease mitigation.

Leukodystrophy caused by plasmalogen deficiency rescued by glyceryl 1-myristyl ether treatment.

Plasmalogens are the most abundant form of ether phospholipids in myelin and their deficiency causes Rhizomelic Chondrodysplasia Punctata (RCDP), a severe developmental disorder. Using the Gnpat-knockout (KO) mouse as a model of RCDP, we determined the consequences of a plasmalogen deficiency during myelination and myelin homeostasis in the central nervous system (CNS). We unraveled that the lack of plasmalogens causes a generalized hypomyelination in several CNS regions including the optic nerve, corpus callosum and spinal cord. The defect in myelin content evolved to a progressive demyelination concomitant with generalized astrocytosis and white matter-selective microgliosis. Oligodendrocyte precursor cells (OPC) and mature oligodendrocytes were abundant in the CNS of Gnpat KO mice during the active period of demyelination. Axonal loss was minimal in plasmalogen-deficient mice, although axonal damage was observed in spinal cords from aged Gnpat KO mice. Characterization of the plasmalogen-deficient myelin identified myelin basic protein and septin 7 as early markers of dysmyelination, whereas myelin-associated glycoprotein was associated with the active demyelination phase. Using in vitro myelination assays, we unraveled that the intrinsic capacity of oligodendrocytes to ensheath and initiate membrane wrapping requires plasmalogens. The defect in plasmalogens was rescued with glyceryl 1-myristyl ether [1-O-tetradecyl glycerol (1-O-TDG)], a novel alternative precursor in the plasmalogen biosynthesis pathway. 1-O-TDG treatment rescued myelination in plasmalogen-deficient oligodendrocytes and in mutant mice. Our results demonstrate the importance of plasmalogens for oligodendrocyte function and myelin assembly, and identified a novel strategy to promote myelination in nervous tissue.

Gene expression analysis in Eucalyptus globulus exposed to drought stress in a controlled and a field environment indicates different strategies for short- and longer-term acclimation.

Previous knowledge suggested the involvement of specific pathways/proteins that could be identified as potential molecular indicators linked to enhanced drought tolerance in Eucalyptus globulus. Here, we looked for specific variations in key transcripts of two Eucalyptus globulus clones (AL-18 and AL-13) exposed to water deficit and rehydration with two main goals: (i) to check if and how transcripts potentially associated with stress response and protection are modulated in a controlled experiment; and (ii) to verify if the transcript response is robust in a field case study. Our results showed that the controlled experiment induced a severe acute stress that resulted in a strong realignment of gene expression resulting from an overwhelming of physiological adjustments to water limitation. A number of transcripts exhibited altered abundance after the acute water stress: reduction of RuBisCO activase and mitochondrial glycine cleavage system H protein, and increase of isoflavone reductase. Malate dehydrogenase, catalase, dehydration response element B1A and potassium channel GORK showed a different abundance pattern in each clone. The stress in the field was more moderate and chronic and the plants were able to deal with the stress primarily through physiological adjustments resulting in much smaller changes in gene expression. The transcripts of clone AL-18 showed few alterations between irrigated and non-irrigated plants throughout the experiment, while the transcript changes found in clone AL-13 highlighted the impact of early rewatering rather than growing under extended drought typical of a Mediterranean summer. Although a few concurrent responses were found, the results obtained in the field study draw a very distinct picture when compared with the controlled experiment.

Combined Drought and Heat Activates Protective Responses in Eucalyptus globulus That Are Not Activated When Subjected to Drought or Heat Stress Alone.

Aiming to mimic a more realistic field condition and to determine convergent and divergent responses of individual stresses in relation to their combination, we explored physiological, biochemical, and metabolomic alterations after drought and heat stress imposition (alone and combined) and recovery, using a drought-tolerant Eucalyptus globulus clone. When plants were exposed to drought alone, the main responses included reduced pre-dawn water potential (Ψpd) and gas exchange. This was accompanied by increases in malondialdehyde (MDA) and total glutathione, indicative of oxidative stress. Abscisic acid (ABA) levels increased while the content of jasmonic acid (JA) fell. Metabolic alterations included reductions in the levels of sugar phosphates accompanied by increases in starch and non-structural carbohydrates. Levels of α-glycerophosphate and shikimate were also reduced while free amino acids increased. On the other hand, heat alone triggered an increase in relative water content (RWC) and Ψpd. Photosynthetic rate and pigments were reduced accompanied by a reduction in water use efficiency. Heat-induced a reduction of salicylic acid (SA) and JA content. Sugar alcohols and several amino acids were enhanced by the heat treatment while starch, fructose-6-phosphate, glucose-6-phosphate, and α-glycerophosphate were reduced. Contrary to what was observed under drought, heat stress activated the shikimic acid pathway. Drought-stressed plants subject to a heat shock exhibited a sharp decrease in gas exchange, Ψpd and JA, no alterations in electrolyte leakage, MDA, starch, and pigments and increased glutathione pool in relation to control. Comparing this with drought stress alone, subjecting drought stressed plants to an additional heat stress alleviated Ψpd and MDA, maintained an increased glutathione pool and reduced starch content and non-structural carbohydrates. A novel response triggered by the combined stress was the accumulation of cinnamate. Regarding recovery, most of the parameters affected by each stress condition reversed after re-establishment of control growing conditions. These results highlight that the combination of drought and heat provides significant protection from more detrimental effects of drought-stressed eucalypts, confirming that combined stress alter plant metabolism in a novel manner that cannot be extrapolated by the sum of the different stresses applied individually.